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Biomedical subjects

Peter R Reeves

Publications and source records attributed to Peter R Reeves.

At least 19 recordsLinked to original sources
PubMed2007

Structural and genetic evidence that the Escherichia coli O148 O antigen is the precursor of the Shigella dysenteriae type 1 O antigen and identification of a glucosyltransferase gene.

Shigella dysenteriae type 1 is the most virulent serotype of Shigella. Enterotoxigenic Escherichia coli O148 is pathogenic and can cause diarrhoea. The following structure was established for the tetrasaccharide repeating unit of the E. coli O148 O antigen: -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpNAc-(1-->. This differs from the structure reported earlier for S. dysenteriae type 1 by having a glucose (Glc) residue in place of a galactose (Gal) residue. The two bacteria also have the same genes for O antigen synthesis, with the same organization and high level of DNA identity, except that in S. dysenteriae type 1 wbbG is interrupted by a deletion, and a galactosyltransferase gene wbbP located on a plasmid is responsible for the transfer of galactose to make a novel antigenic epitope of the O antigen. The S. dysenteriae type 1 O antigen was reconstructed by replacing the E. coli O148 wbbG gene with the wbbP gene, and it had the LPS structure and antigenic properties of S. dysenteriae type 1, indicating that the S. dysenteriae type 1 O antigen evolved from that of E. coli O148. It was also confirmed that wbbG of E. coli O148 is a glucosyltransferase gene, and two serotype-specific genes of E. coli O148 and S. dysenteriae type 1 were identified.

Bacterial Proteins↗
PubMed2006

Molecular markers with potential to replace phage typing for Salmonella enterica serovar typhimurium.

Using Amplified Fragment Length Polymorphism (AFLP) analysis of isolates from 23 phage types, we isolated 11 molecular markers that are potentially useful for molecular typing of Salmonella enterica serovar typhimurium. We tested these and 11 previously studied markers for their ability to discriminate among isolates and for correlation of their distribution with phage types. The Simpson's index of discriminatory power for the molecular markers is 0.96. One hundred and twenty one isolates from 33 phage types tested were divided into 51 types which are further grouped into 24 patterns. Eight patterns can unambiguously identify 8 phage types and a further 12 correlated with phage type distribution, showing the usefulness of these markers for molecular phage typing.

Bacteriophage Typing↗
PubMed2006

Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes.

Several major invasive bacterial pathogens are encapsulated. Expression of a polysaccharide capsule is essential for survival in the blood, and thus for virulence, but also is a target for host antibodies and the basis for effective vaccines. Encapsulated species typically exhibit antigenic variation and express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. We provide the sequences of the capsular biosynthetic genes of all 90 serotypes of Streptococcus pneumoniae and relate these to the known polysaccharide structures and patterns of immunological reactivity of typing sera, thereby providing the most complete understanding of the genetics and origins of bacterial polysaccharide diversity, laying the foundations for molecular serotyping. This is the first time, to our knowledge, that a complete repertoire of capsular biosynthetic genes has been available, enabling a holistic analysis of a bacterial polysaccharide biosynthesis system. Remarkably, the total size of alternative coding DNA at this one locus exceeds 1.8 Mbp, almost equivalent to the entire S. pneumoniae chromosomal complement.

Bacterial Capsules↗
PubMed2006

Sex and virulence in Escherichia coli: an evolutionary perspective.

Pathogenic Escherichia coli cause over 160 million cases of dysentery and one million deaths per year, whereas non-pathogenic E. coli constitute part of the normal intestinal flora of healthy mammals and birds. The evolutionary pathways underlying this dichotomy in bacterial lifestyle were investigated by multilocus sequence typing of a global collection of isolates. Specific pathogen types [enterohaemorrhagic E. coli, enteropathogenic E. coli, enteroinvasive E. coli, K1 and Shigella] have arisen independently and repeatedly in several lineages, whereas other lineages contain only few pathogens. Rates of evolution have accelerated in pathogenic lineages, culminating in highly virulent organisms whose genomic contents are altered frequently by increased rates of homologous recombination; thus, the evolution of virulence is linked to bacterial sex. This long-term pattern of evolution was observed in genes distributed throughout the genome, and thereby is the likely result of episodic selection for strains that can escape the host immune response.

Alleles↗
PubMed2005

Adaptation of multilocus sequencing for studying variation within a major clone: evolutionary relationships of Salmonella enterica serovar Typhimurium.

Serovar Typhimurium of Salmonella enterica is a model organism for studies of pathogenesis that exhibits phage-type variation and variation in host range and virulence, but in a recent study showed no sequence variation in four genes, indicating the clonal nature of this serovar. We determined the relationships of 46 Typhimurium isolates of nine phage types using mutational changes detected either by matching AFLP (amplified fragment length polymorphism) fragments to computer-modeled LT2 AFLP fragments or by sequencing intergenic regions. Fifty-one polymorphic sites were detected, which gave a single phylogenetic tree. Comparison with genome sequences of five other serovars, Typhi, Paratyphi A, Gallinarum, Enteritidis, and Pullorum, enabled determination of the root of the tree. Only two parallel events were observed, giving high confidence in the tree branching order. The mutation-based tree provided a high level of consistency and a clear lineage for the Typhimurium isolates studied. This enabled us to show that for seven of the nine phage types used, the isolates studied have a single origin, but that two phage types clearly have more than one independent origin. We found that sequencing intergenic regions provides a good strategy for detection of mutational polymorphisms and study of phylogenetic relationships of closely related isolates and would be applicable to many other species.

Clone Cells↗
PubMed2005

Evolutionary origins and sequence of the Escherichia coli O4 O-antigen gene cluster.

Escherichia coli express many types of O antigen, present in the outer membrane of the Gram-negative bacterial cell wall. O-Antigen biosynthesis genes are clustered together and differences seen in O-antigen types are due to genetic variation within this gene cluster. Sequencing of the E. coli O4 O-antigen gene cluster revealed a similar gene order and high levels of similarity to that of E. coli O26; indicating a common ancestor. These lateral transfer events observed within O-antigen gene clusters may occur as part of the evolution of the pathogenic clones.

Base Sequence↗
PubMed2005

Evolutionary changes of the flhDC flagellar master operon in Shigella strains.

Shigella strains are nonmotile. The master operon of flagellar synthesis, flhDC, was analyzed for genetic damage in 46 Shigella strains representing all known serotypes. In 11 strains (B1, B3, B6, B8, B10, B18, D5, F1B, D10, F3A, and F3C) the flhDC operon was completely deleted. PCR and sequence analysis of the flhDC region of the remaining 35 strains revealed many insertions or deletions associated with insertion sequences, and the majority of the strains were found to be defective in their flhDC genes. As these genes also play a role in regulation of non-flagellar genes, the loss may have other consequences or be driven by selection pressures other than those against flagellar motility. It has been suggested that Shigella strains fall mostly into three clusters within Escherichia coli, with five outlier strains, four of which are also within E. coli (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000). The distribution of genetic changes in the flhDC region correlated very well with the three clusters and outlier strains found using housekeeping gene DNA sequences, enabling us to follow the sequence of mutational change in the flhDC locus. Two cluster 2 strains were found to have unique flhDC sequences, which are most probably due to recombination during the exchange of the adjacent O-antigen gene clusters.

Base Sequence↗
PubMed2005

Structural and genetic characterization of enterohemorrhagic Escherichia coli O145 O antigen and development of an O145 serogroup-specific PCR assay.

Enterohemorrhagic Escherichia coli O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the E. coli O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamidoylamino-2,6-dideoxy-L-galactose, and N-acetylneuraminic acid. It is very closely related to Salmonella enterica serovar Touera and S. enterica subsp. arizonae O21 antigen. The E. coli O145 gene cluster is located between the JUMPStart sequence and the gnd gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the E. coli O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An E. coli O145 serogroup-specific PCR assay based on the genes wzx and wzy was also developed by screening E. coli and Shigella isolates of different serotypes.

Amino Sugars↗
PubMed2005

Structural and genetic characterization of the Shigella boydii type 10 and type 6 O antigens.

Comparison of the O antigens of Shigella boydii types 10 and 6 by chemical analysis and nuclear magnetic resonance spectroscopy showed that their structures are similar, with the only difference being the presence or absence of d-ribofuranose, which is the immunodominant sugar in S. boydii type 10. In S. boydii type 6, a residue previously reported as alpha-d-GlcpA, was shown to be beta-d-GlcpA as in S. boydii type 10. S. boydii types 10 and 6 are reported not to cross-react serologically, and the role of d-ribofuranose in the specificity of S. boydii was confirmed by making a mutant of type 10 that lacked d-ribofuranose. However, S. boydii type 11, which has a d-ribofuranose but with different linkage does show cross-reaction with type 10. The O-antigen gene loci of S. boydii types 10 and 6 were shown to be virtually identical except that orf8 (wbaM), which was confirmed as the ribofuranosyltransferase gene, is interrupted by IS629 in type 6. Therefore, it is proposed that the O-antigen gene cluster of S. boydii type 6 was derived from type 10 by an IS element insertion.

Amino Acid Sequence↗
PubMed2005

Vibrio cholerae pathogenic clones.

We resolved the relationships between 2 pandemic clones of Vibrio cholerae. Using 26 housekeeping genes, we showed that the US Gulf clone, the Australian clone, and 3 El Tor strains isolated before the seventh pandemic were related to the seventh pandemic clone. The sixth pandemic clone was well separated from them.

Bacterial Proteins↗
PubMed2004

Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp.

Enteroinvasive Escherichia coli (EIEC), a distinctive pathogenic form of E. coli causing dysentery, is similar in many properties to bacteria placed in the four species of Shigella. Shigella has been separated as a genus but in fact comprises several clones of E. coli. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigella strains. The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times. Three of the four clusters contain more than one O antigen type. One EIEC strain (an O112ac:H- strain) was found in Shigella cluster 3 but is not identical to the Shigella cluster 3 D2 and B15 strains with the same O antigen. Two forms of the virulence plasmid pINV have been identified in Shigella strains by using the sequences of ipgD and mxiA genes, and all but two of our EIEC strains have pINV A. The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with Shigella pINV A strains. The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions. Two strains were found in the pINV B form by using the ipgD sequence, with one strain having an mxiA sequence similar to the divergent sequence of D1. Clearly, EIEC and Shigella spp. form a pathovar of E. coli.

Bacterial Proteins↗
PubMed2004

Synthesis of the heteropolysaccharide O antigen of Escherichia coli O52 requires an ABC transporter: structural and genetic evidence.

The structural and genetic organization of the Escherichia coli O52 O antigen was studied. As identified by sugar and methylation analysis and nuclear magnetic resonance spectroscopy, the O antigen of E. coli O52 has a partially O-acetylated disaccharide repeating unit (O unit) containing D-fucofuranose and 6-deoxy-D-manno-heptopyranose, as well as a minor 6-deoxy-3-O-methylhexose (most likely, 3-O-methylfucose). The O-antigen gene cluster of E. coli O52, which is located between the galF and gnd genes, was found to contain putative genes for the synthesis of the O-antigen constituents, sugar transferase genes, and ABC-2 transporter genes. Further analysis confirmed that O52 employs an ATP-binding cassette (ABC) transporter-dependent pathway for translocation and polymerization of the O unit. This is the first report of an ABC transporter being involved in translocation of a heteropolysaccharide O antigen in E. coli. Genes specific for E. coli O52 were also identified.

ATP-Binding Cassette Transporters↗
PubMed2004

Relationships of the Escherichia coli O157, O111, and O55 O-antigen gene clusters with those of Salmonella enterica and Citrobacter freundii, which express identical O antigens.

Escherichia coli O157, Salmonella enterica O30, and Citrobacter freundii F90 have identical O-antigen structures, as do E. coli O55 and S. enterica O50. The O-antigen gene cluster sequences for E. coli O157 and E. coli O55 have been published, and the genes necessary for O-antigen biosynthesis have been identified, although transferase genes for glycosidic linkages are only generic and have not been allocated to specific linkages. We determined sequences for S. enterica O30 and C. freundii F90 O-antigen gene clusters and compared them to the sequence of the previously described E. coli O157 cluster. We also determined the sequence of the S. enterica O50 O-antigen gene cluster and compared it to the sequence of the previously described E. coli O55 cluster. For both the S. enterica O30-C. freundii F90-E. coli O157 group and the S. enterica O50-E. coli O55 group of O antigens, the gene clusters have identical or nearly identical organizations. The two sets of gene clusters had comparable overall levels of similarity in their genes, which were lower than the levels determined for housekeeping genes for these species, which were 55 to 65% for the genes encoding glycosyltransferases and O-antigen processing proteins and 75 to 93% for the nucleotide-sugar pathway genes. Nonetheless, the similarity of the levels of divergence in the five gene clusters required us to consider the possibility that the parent gene cluster for each structure was in the common ancestor of the species and that divergence is faster than expected for the common ancestor hypothesis. We propose that the identical O-antigen gene clusters originated from a common ancestor, and we discuss some possible explanations for the increased rate of divergence that is seen in these genes.

Base Sequence↗
PubMed2004

Molecular basis of the indole-negative reaction in Shigella strains: extensive damages to the tna operon by insertion sequences.

The molecular basis of the loss of tryptophan utilization (indole-negative phenotype) of Shigella strains, in effect clones of Escherichia coli, was investigated. Analysis of the tna operon of 23 Shigella strains representing each of the indole-negative serotypes revealed that insertion sequence-mediated insertion and/or deletions damaged the tna operon, leading to inability to convert tryptophan to indole. These events differ for cluster 1, cluster 3, and the outlier Shigella strains, confirming our previous observation of independent origins of these lineages from within E. coli. Parallel loss of the trait and prevalence of indole-negative strains suggest that the trait is deleterious in Shigella strains and advantages those without it.

DNA Transposable Elements↗
PubMed2004

Deletion of the Escherichia coli O14:K7 O antigen gene cluster.

Escherichia coli O14:K7 is a rough strain, lacking a typical O antigen, in which the enterobacterial common antigen is attached to the lipopolysaccharide core. The rough phenotype was previously mapped to the O antigen gene cluster; however, the nature of the nonfunctional locus was not defined. In this study, we have shown that the O antigen gene cluster of an O14:K7 type strain (Su4411/41) was most likely deleted via homologous recombination between the GDP-mannose pathway genes (manB and manC) of the colanic acid and O antigen gene clusters. A similar recombination event has previously been inferred for the deletion of E. coli Sonnei chromosomal O antigen genes. Therefore, recombination between the GDP-mannose pathway genes provides a convenient mechanism for the deletion of O antigen genes, which may occur if the typical O antigen becomes redundant.

Escherichia coli↗
PubMed2003

AFLP analysis of Salmonella enterica serovar Typhimurium isolates of phage types DT 9 and DT 135: diversity within phage types and its epidemiological significance.

Amplified fragment length polymorphism (AFLP) was applied to 35 and 34 isolates, respectively, of Salmonella enterica serovar Typhimurium phage types DT 9 and DT 135, using eight primer pair combinations. Eight and 17 AFLP types were observed in DT 9 and DT 135, respectively. DT 9 is rare in the UK and common in Australia, but one AFLP form dominated with 28 isolates, comprising 22 of 25 UK isolates, four of five Australian isolates, one Jamaican and one Spanish isolate. Of the others, two UK isolates are closely related to the major form, two from elsewhere are in the major cluster and three isolates from different countries are in a separate cluster. For DT 135, two closely related AFLP types of seven and 11 isolates form the major cluster, which also includes 11 isolates, mostly in single-isolate AFLP types, while five isolates from different countries form a well-separated minor cluster. For both DTs all isolates are grouped together if only the phage type specific bands identified earlier are used, confirming their value for molecular-based 'phage typing'. Polymorphic markers identified in this study could also be used for subtyping within both phage types. The value of AFLP is in locating DNA fragments useful for typing, but implementation of a replacement typing scheme would probably involve multiplex PCR or microarray technologies.

Bacterial Typing Techniques↗
PubMed2003

The variation of dTDP-L-rhamnose pathway genes in Vibrio cholerae.

The genetic variation in the dTDP-L-rhamnose pathway genes (rmlA, rmlB, rmlC and rmlD) in Vibrio cholerae was investigated. The genes are part of the O antigen gene cluster and the aim was to study lateral gene transfer of O antigen gene clusters. The rml genes of an O6 strain were cloned using an Escherichia coli K-12 strain designed for selecting cloned rml genes. Thirty-three strains carrying the known rhamnose-containing O antigens were probed with O6-based rml gene probes, and 19 were positive with from one to all four of the gene probes. Nine rml gene sets from this group were sequenced and found to be in the order rmlBADC, at the 5' end of the gene clusters. A gradient in the level of variation was observed, with highly similar sequences at the 5' end rmlB gene, but very divergent and strain-specific sequences at the 3' end of the rml gene set. The change in level of similarity varied in position, but was always abrupt and coincided with a change in GC content, indicating that the 5' and 3' parts are of different origin, and that recombination within rml genes has occurred. The rml gene sets of two of the strains that did not hybridize with any O6 rml gene probes were also cloned and sequenced. Both gene sets were in the middle of the O antigen gene cluster and were very divergent from each other and all other rml gene sets. This supports the hypothesis that presence of rml genes at the end of the O antigen gene cluster facilitates lateral gene transfer of rml-containing O antigen gene clusters in V. cholerae. The sequence relationships make it possible to identify sites of recombination and to distinguish DNA that has long been in V. cholerae and DNA that probably came into the species with the O antigen gene cluster.

Amino Acid Sequence↗